Bottom Line:
We found that PKP3 binds all three desmogleins, desmocollin (Dsc) 3a and -3b, and possibly also Dsc1a and -2a.Evidence was found for the presence of at least two DP-PKP3 interaction sites.Together, these results show that PKP3, whose epithelial and epidermal desmosomal expression pattern and protein interaction repertoire are broader than those of PKP1 and -2, is a unique multiprotein binding element in the basic architecture of a vast majority of epithelial desmosomes.

ABSTRACTPlakophilin 3 (PKP3) is a recently described armadillo protein of the desmosomal plaque, which is synthesized in simple and stratified epithelia. We investigated the localization pattern of endogenous and exogenous PKP3 and fragments thereof. The desmosomal binding properties of PKP3 were determined using yeast two-hybrid, coimmunoprecipitation and colocalization experiments. To this end, novel mouse anti-PKP3 mAbs were generated. We found that PKP3 binds all three desmogleins, desmocollin (Dsc) 3a and -3b, and possibly also Dsc1a and -2a. As such, this is the first protein interaction ever observed with a Dsc-b isoform. Moreover, we determined that PKP3 interacts with plakoglobin, desmoplakin (DP) and the epithelial keratin 18. Evidence was found for the presence of at least two DP-PKP3 interaction sites. This finding might explain how lateral DP-PKP interactions are established in the upper layers of stratified epithelia, increasing the size of the desmosome and the number of anchoring points available for keratins. Together, these results show that PKP3, whose epithelial and epidermal desmosomal expression pattern and protein interaction repertoire are broader than those of PKP1 and -2, is a unique multiprotein binding element in the basic architecture of a vast majority of epithelial desmosomes.

fig10: Clustal W alignment of the PKP3 protein orthologues from man (hsPKP3), mouse (mmPKP3), rat (rnPKP3), and X. laevis (xlPKP3) shaded using the BOXSHADE server (http://www.ch.embnet.org). Only those aa are shaded that are identical (black) or similar (gray) in each of the sequences. The HR2 domain is indicated by the top line, and the start of the arm domains is indicated by an arrow. The general sequence conservation in the arm domains is striking compared with the situation in the head domains, where only short sequence stretches are conserved. Database accession nos. are: AF053719 (hsPKP3), AF136719 (mmPKP3), and AX046097 (xlPKP3). The rat PKP3 protein sequence was predicted on the basis of the genomic sequences identified by BLAT search at http://genome.ucsc.edu/goldenPath (UCSC Rat Genome Project, November 2002 release).

Mentions:
By use of the yeast two-hybrid system, we analyzed direct desmosomal PKP3 interactions and observed clear-cut PKP3 interactions with all desmosomal cadherins but Dsc1b and Dsc2b. The PKP3 protein domain that interacts with Dsg1 was confined to the nonarmadillo head domain of the protein, as has also been reported for the Dsg1–PKP1 and Dsg1–PKP2 interactions (Smith and Fuchs, 1998; Kowalczyk et al., 1999a; Hatzfeld et al., 2000; Chen et al., 2002). The exact sequence of the PKP3head domain responsible for this interaction is still unknown. For PKP1, it has been reported that aa 70–213, residing in the head domain of PKP1, are sufficient to interact with Dsg1 (Hatzfeld et al., 2000). Removal of the single conserved sequence stretch in the PKP head domains, i.e., the HR2 domain involved in DP binding, had no effect on Dsg1 binding in the yeast two-hybrid system. In contrast, several conserved sequence stretches can be observed in the head domains of the human, mouse, rat, and Xenopus laevis PKP3 orthologues, which might include PKP3-specific Dsg1 interaction sites (Fig. 10) . Using Dsg1 deletion constructs, we found two physically separable PKP3 interaction sites (namely the IA+CBS and the Dsg domain), whereas PKP1 was previously reported to bind the CBS+Dsg domain (Hatzfeld et al., 2000). Hence, it appears that PKP1 and PKP3 head domains bind different regions of the Dsg1 intracellular domain. Also in the Dsg2 tail, two separable PKP3 interaction sites were identified by us. The PKP3 interactions observed in the yeast two-hybrid system were confirmed, where possible, in CoIP and colocalization experiments. These experiments further strengthened the evidence for a direct interaction between PKP3, and Dsg1 to -3, Dsc1a, Dsc2a, Dsc3a, and Dsc3b. Although it was less clear from yeast two-hybrid experiments whether PKP3 and Dsc3a or Dsc3b interact, strong evidence for such interactions was provided by CoIP experiments. Moreover, the PKP3 binding site in the Dsc3a/Dsc3b cytoplasmic tails could be narrowed down to 36 membrane-proximal aa. As such, PKP3 is the first protein interaction partner of a Dsc-b isoform ever identified.

fig10: Clustal W alignment of the PKP3 protein orthologues from man (hsPKP3), mouse (mmPKP3), rat (rnPKP3), and X. laevis (xlPKP3) shaded using the BOXSHADE server (http://www.ch.embnet.org). Only those aa are shaded that are identical (black) or similar (gray) in each of the sequences. The HR2 domain is indicated by the top line, and the start of the arm domains is indicated by an arrow. The general sequence conservation in the arm domains is striking compared with the situation in the head domains, where only short sequence stretches are conserved. Database accession nos. are: AF053719 (hsPKP3), AF136719 (mmPKP3), and AX046097 (xlPKP3). The rat PKP3 protein sequence was predicted on the basis of the genomic sequences identified by BLAT search at http://genome.ucsc.edu/goldenPath (UCSC Rat Genome Project, November 2002 release).

Mentions:
By use of the yeast two-hybrid system, we analyzed direct desmosomal PKP3 interactions and observed clear-cut PKP3 interactions with all desmosomal cadherins but Dsc1b and Dsc2b. The PKP3 protein domain that interacts with Dsg1 was confined to the nonarmadillo head domain of the protein, as has also been reported for the Dsg1–PKP1 and Dsg1–PKP2 interactions (Smith and Fuchs, 1998; Kowalczyk et al., 1999a; Hatzfeld et al., 2000; Chen et al., 2002). The exact sequence of the PKP3head domain responsible for this interaction is still unknown. For PKP1, it has been reported that aa 70–213, residing in the head domain of PKP1, are sufficient to interact with Dsg1 (Hatzfeld et al., 2000). Removal of the single conserved sequence stretch in the PKP head domains, i.e., the HR2 domain involved in DP binding, had no effect on Dsg1 binding in the yeast two-hybrid system. In contrast, several conserved sequence stretches can be observed in the head domains of the human, mouse, rat, and Xenopus laevis PKP3 orthologues, which might include PKP3-specific Dsg1 interaction sites (Fig. 10) . Using Dsg1 deletion constructs, we found two physically separable PKP3 interaction sites (namely the IA+CBS and the Dsg domain), whereas PKP1 was previously reported to bind the CBS+Dsg domain (Hatzfeld et al., 2000). Hence, it appears that PKP1 and PKP3 head domains bind different regions of the Dsg1 intracellular domain. Also in the Dsg2 tail, two separable PKP3 interaction sites were identified by us. The PKP3 interactions observed in the yeast two-hybrid system were confirmed, where possible, in CoIP and colocalization experiments. These experiments further strengthened the evidence for a direct interaction between PKP3, and Dsg1 to -3, Dsc1a, Dsc2a, Dsc3a, and Dsc3b. Although it was less clear from yeast two-hybrid experiments whether PKP3 and Dsc3a or Dsc3b interact, strong evidence for such interactions was provided by CoIP experiments. Moreover, the PKP3 binding site in the Dsc3a/Dsc3b cytoplasmic tails could be narrowed down to 36 membrane-proximal aa. As such, PKP3 is the first protein interaction partner of a Dsc-b isoform ever identified.

Bottom Line:
We found that PKP3 binds all three desmogleins, desmocollin (Dsc) 3a and -3b, and possibly also Dsc1a and -2a.Evidence was found for the presence of at least two DP-PKP3 interaction sites.Together, these results show that PKP3, whose epithelial and epidermal desmosomal expression pattern and protein interaction repertoire are broader than those of PKP1 and -2, is a unique multiprotein binding element in the basic architecture of a vast majority of epithelial desmosomes.

ABSTRACTPlakophilin 3 (PKP3) is a recently described armadillo protein of the desmosomal plaque, which is synthesized in simple and stratified epithelia. We investigated the localization pattern of endogenous and exogenous PKP3 and fragments thereof. The desmosomal binding properties of PKP3 were determined using yeast two-hybrid, coimmunoprecipitation and colocalization experiments. To this end, novel mouse anti-PKP3 mAbs were generated. We found that PKP3 binds all three desmogleins, desmocollin (Dsc) 3a and -3b, and possibly also Dsc1a and -2a. As such, this is the first protein interaction ever observed with a Dsc-b isoform. Moreover, we determined that PKP3 interacts with plakoglobin, desmoplakin (DP) and the epithelial keratin 18. Evidence was found for the presence of at least two DP-PKP3 interaction sites. This finding might explain how lateral DP-PKP interactions are established in the upper layers of stratified epithelia, increasing the size of the desmosome and the number of anchoring points available for keratins. Together, these results show that PKP3, whose epithelial and epidermal desmosomal expression pattern and protein interaction repertoire are broader than those of PKP1 and -2, is a unique multiprotein binding element in the basic architecture of a vast majority of epithelial desmosomes.